GIPZ™ Lentiviral shRNA

GIPZ

™

Lentiviral shRNA

TECHNICAL MANUAL

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Product description

The Dharmacon

™

GIPZ

™

Lentiviral shRNA Library was developed in collabora-

tion with Dr. Greg Hannon of Cold Spring Harbor Laboratory [CSHL] and Dr.

Steve Elledge of Harvard Medical School. This library combines the design

advantages of microRNA-adapted shRNA with the pGIPZ lentiviral vector to

create a powerful RNA tool capable of producing RNA interference (RNAi) in

most cell types including primary and non-dividing cells.

Important safety note

Please follow the safety guidelines for use and production of vector-based

lentivirus as set by your institution’s biosafety committee.

• For glycerol stocks of E. coli containing lentiviral plasmids, BSL1 guidelines

should be followed

• For handling and use of lentiviral products to produce lentiviral particles,

BSL2 or BSL2+ guidelines should be followed

• For handling and use of lentiviral particle products, BSL2 or BSL2+ guide-

lines should be followed

Additional information on the safety features incorporated in the pGIPZ

lentiviral vector and the Horizon Trans-Lentiviral Packaging System can be

found on page 3.

Design information

Unique microRNA-30 based hairpin design

Short hairpin RNA (shRNA) constructs are expressed as human microRNA-30

(miR-30) primary transcripts. This design adds a Drosha processing site to the

hairpin construct and has been shown to greatly increase gene silencing

eciency (Boden 2004). The hairpin stem consists of 22 nucleotides (nt) of

dsRNA and a 19 nucleotides (nt) loop from human miR-30. Adding the miR-30

loop and 125 nucleotides (nt) of miR-30 anking sequence on either side of the

hairpin results in greater than 10-fold increase in Drosha and Dicer processing

of the expressed hairpins when compared with conventional shRNA designs

(Silva 2005). Increased Drosha and Dicer processing translates into greater

shRNA production and greater potency for expressed hairpins.

Use of the miR-30 design also allowed the use of ‘rules-based’ designs for target

sequence selection. One such rule is the destabilizing of the 5' end of the

antisense strand, which results in strand specic incorporation of microRNA/

siRNAs into RISC. The proprietary design algorithm targets sequences in coding

regions and the 3' UTR with the additional requirement that they contain great-

er than 3 mismatches to any other sequence in the human or mouse genomes.

Each shRNA construct has been bioinformatically veried to match NCBI

sequence data. To assure the highest possibility of modulating the gene

expression level, each gene is represented by multiple shRNA constructs, each

covering a unique region of the target gene.

Vector information

Versatile vector design

Features of the pGIPZ lentiviral vector (Figures 1 and 2) that make it a versatile

tool for RNAi studies include:

• Ability to perform transfections or transductions using the replication

incompetent lentivirus (Shimada 1995)

• TurboGFP™ (Evrogen, Moscow, Russia) and shRNA are part of a bicistronic

transcript allowing the visual marking of shRNA expressing cells.

• Amenable to in vitro and in vivo applications.

• Puromycin drug resistance marker for selecting stable cell lines.

• Molecular barcodes enable multiplexed screening in pools

Dharmacon

™

GIPZ

™

shRNA vectors are not compatible with

third generation packaging systems, due to the requirement

of the expression of tat, which third generation systems do

not contain. We recommend the Trans-Lentiviral Packaging

System for use with our vectors.

GIPZ shRNA is available in glycerol stock or viral particle

format. If viral particle format is purchased, begin work with

Protocol IX – Determining Relative Transduction Efficiency.

pGIPZ

11.8 kb

Drawing was created using Geneious (http://www.geneious.com).

Vector map

Figure 2. Detailed vector map of pGIPZ lentiviral vector.

Antibiotic resistance

pGIPZ contains three antibiotic resistance markers (Table 2).

Puro

IRES

Amp

SV40 ori

pUC ori

tGFP

hCMV

RRE

shRNA

WPRE

5' LTR

3' SIN LTR

pGIPZ

pTRIPZ

rtTA3

tRFP

TRE

UBC

From PowerPoint (R&D)Revised versions

pLOC

pLKO.1

hPGK

pSMART 2.0

SV40 ori

hCMV

pSMART 2.0

Amp

pUCori

SV40 ori

5ʹ LTR

3ʹ SIN LTR

RRE

tGFP

hCMV

IRES

Puro

shRNA WPRE

cPPT/CTS

hPGK

Amp

pUCori

pLKO.1

RSV/5ʹ LTR

3ʹ SIN LTR

RRE

Puro

shRNA

pSMART 2.0

Amp

pUCori

SV40 ori

5ʹ LTR

3ʹ SIN LTR

RRE

tGFP

hCMV

IRES

Puro

microRNA

WPRE

pLOC

Amp

pUCori

SV40 ori

5ʹ LTR

3ʹ SIN LTR

RRE

tGFP

nuc

hCMV

IRES

Blast

-2a-

WPRE

ORF

Multi Tag

Cloning Site

Amp

pUCori

SV40 ori

pGIPZ

5ʹ LTR

3ʹ SIN LTR

RRE

tGFP

hCMV

IRES

Puro

WPRE

shRNA

Amp

pUCori

SV40 ori

UBC

pTRIPZ

5ʹ LTR

3ʹ SIN LTR

RRE

tRFP

TRE

IRES

Puro

WPRE

rtTA3

shRNA

hCMV

mCMV

hEF1α

mEF1α

CAG

PGK

UBC

SMARTchoiceshRNA

5ʹ LTR

3ʹ SIN LTR

RRE

tGFP

tRFP

IRES

Puro

WPRE

SMARTc hoice

promoters

SMARTvector2.0 universal scaold

5' LTR

Puro

IRES

WPRE

RRE

shRNA

3' SIN LTR

Amp

SV40 ori

pUC ori

hCMV

5' LTR

RSV/5' LTR

Puro

IRES

WPRE

RRE

shRNA

3' SIN LTR

5' LTR

3' SIN LTR

Amp

SV40 ori

pUC ori

SV40 ori

Amp

pUC ori

tGFP

Puro

IRES

WPRE

microRNA

tGFP

hCMV

Blast

IRES

WPRE

-2a-

tGFP

nuc

Multi Tag

Cloning Site

ORF

hCMV

mCMV

hEF1

mEF1α

CAG

PGK

UBC

SMARTchoice

promoters

5' LTR

tGFP

Puro

IRES

tRFP

WPRE

3' SIN LTR

SMARTvector universal scaold

SMARTchoice shRNA

RRE

Vector element Utility

hCMV Human cytomegalovirus promoter drives strong transgene

expression

tGFP TurboGFP reporter for visual tracking of transduction and

expression

Puro

Puromycin resistance permits antibiotic-selective pressure

and propagation of stable integrants

IRES Internal ribosomal entry site allows expression of TurboGFP

and puromycin resistance genes in a single transcript

shRNA microRNA-adapted shRNA (based on miR-30) for

gene knockdown

5' LTR 5' long terminal repeat

3' SIN LTR 3' self-inactivating long terminal repeat for increased

lentivirus safety

Psi packaging sequence allows viral genome packaging

using lentiviral packaging systems

RRE Rev response element enhances titer by increasing

packaging efficiency of full-length viral genomes

WPRE Woodchuck hepatitis posttranscriptional regulatory

element enhances transgene expression in the target cells

Table 1. Features of the pGIPZ vector.

Figure 1. pGIPZ lentiviral vector.

Antibiotic Concentration Utility

Ampicillin (carbenicillin) 100 μg/mL Bacterial selection marker

(outside LTRs)

Zeocin

™

25 μg/mL Bacterial selection marker

(inside LTRs)

Puromycin Variable Mammalian selection marker

Table 2. Antibiotic resistances conveyed by pGIPZ.

Quality control

The GIPZ Lentiviral shRNA Library has passed through internal QC processes to

ensure high quality and low recombination (Figures 3 and 4).

Figure 3. Representative GIPZ Lentiviral shRNA clones grown for 16 hours at 30 °C.

Plasmid was isolated and normalized to a standard concentration. Clones were then

digested with SacII and run on an agarose gel with uncut plasmid. The expected band

sizes are 1259 bp, 2502 bp, and 7927 bp. No recombinant products are visible. 10 kb

molecular weight ladder (10 kb, 7 kb, 5 kb, 4 kb, 3 kb, 2.5 kb, 2 kb, 1.5 kb, 1 kb).

Figure 4. Gel image of a restriction digest of clones from the GIPZ shRNA library

cultured for 10 successive generations in an attempt to determine the tendency of the

pGIPZ vector to recombine. Each generation was thawed, replicated and incubated

overnight for 16 hours at 30 °C then frozen, thawed and replicated. This process was

repeated for 10 growth cycles. After the 10th growth cycle, plasmid was isolated and

normalized to a standard concentration. Clones were digested with SacII and run on

an agarose gel. The pGIPZ vector appears stable without showing any recombination.

Expected band sizes: 1259 bp, 2502 bp, and 7927 bp. 10 kb molecular weight ladder

(10 kb, 7 kb, 5 kb, 4 kb, 3 kb, 2.5 kb, 2 kb, 1.5 kb, 1 kb).

Additional safety information

Historically, the greatest safety risk associated with a lentiviral delivery

platform stems from the potential generation of recombinant viruses that

are capable of autonomous replication. The pGIPZ Lentiviral shRNA platform

minimizes these hazards to the greatest degree by combining a disabled viral

genome with the proprietary Trans-Lentiviral packaging process. Starting with

the HXB2 clone of HIV-1 (GenBank, Accession #K03455), the lentiviral backbone

has been modied to eliminate all but the most essential genetic elements

necessary for packaging and integration (such as 5' LTR, Psi sequences, polypu-

rine tracts, Rev responsive elements and 3' LTR). The resultant self-inactivating

(SIN) vector greatly reduces the probability of producing recombinant particles

and limits cellular toxicity often associated with expression of HIV genes.

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Additional safety features can be incorporated by the packaging process

itself. Generation of pGIPZ Lentiviral shRNA particles requires a packaging

step during which the expression construct containing the silencing sequence

is enclosed in a viral capsid. Gene functions that facilitate this process (such

as those encoded by the structural genes gag, pol, env, etc.) are distributed

amongst multiple helper plasmids which do not contain signicant regions

of homology. This tactic further minimizes the probability of recombination

events that might otherwise generate viruses capable of autonomous

replication. Among commercially available lentiviral vector systems, the

Trans-Lentiviral Packaging System oers a superior safety prole as the

packaging components are separated onto ve plasmids. Additionally,

expression of gag-pro and tat-rev are under the control of the conditional

tetracycline-responsive promoter element (TRE), limiting expression of

these viral components strictly to the packaging cell line. A detailed description

of the Trans-Lentiviral Packaging System can be found in (Wu 2000).

With these safety measures in place, GIPZ lentiviral shRNA particles can be

employed in standard Biosafety Level 2 tissue culture facilities.

Any investigator who purchases Horizon viral vector products is responsible

for consulting with their institution’s health and biosafety group for specic

guidelines on the handling of lentiviral vector particles. Further, each

investigator is fully responsible for obtaining the required permissions for the

acceptance of lentiviral particles into their local geography and institution.

• In the U.S., download the U.S. Department of Health and Human Services

Centers for Disease Control and Prevention and National Institutes of

Health, Biosafety in Microbiological and Biomedical Laboratories (BMBL),

Fifth Edition, Feb 2007 here.

• See also: NIH Guidelines For Research Involving Recombinant DNA

Molecules (NIH Guidelines), September 2009, downloadable here.

• For Biosafety Considerations for Research with Lentiviral Vectors, see.

Replication of individual clones

Once the clone has been streak isolated and the identity of the strain has been

conrmed** by Sanger sequencing (See: What is the sequencing primer for

pGIPZ?), we recommend making a stock of the pure culture. Grow the pure

culture in LB broth + appropriate antibiotic (See protocol below: Protocol

1 - replication). Vortex the culture to evenly mix the glycerol throughout the

culture. The culture can be stored indenitely at –80 °C.

**Testing of 3-5 colonies is recommended.

Protocol I – replication

For archive replication, grow GIPZ shRNA clones at 30 °C in 2x LB broth

(low salt)* medium plus 25 μg/mL Zeocin

™

and 100 μg/mL carbenicillin in order

to provide maximum stability of the clones. Prepare medium with 8% glycerol**

and the appropriate antibiotics.

2x LB broth (low-salt) medium preparation

LB-Broth-Lennox 20 g/L

Peptone 10 g/L

Yeast Extract 5 g/L

Appropriate antibiotic(s) at recommended concentration(s). Glycerol 8% for

long-term storage.

*1x LB medium can be used instead of 2x LB broth medium.

**Glycerol can be omitted from the medium if you are culturing for plasmid

preparation. If making copies of the constructs for long-term storage at

–80 °C, 8% glycerol is required

Table 3. Materials for plate replication.

Item Vendor Cat #

2x LB Broth (low salt) Fisher Scientific BP1427500

Peptone, granulated, 2 kg – Difco Fisher Scientific BP9725-2

Yeast Extract, 500 g, granulated Fisher Scientific BP1422-500

NaCl Fisher Scientific BP3581

Glycerol Fisher Scientific BP2291

Carbenicillin Fisher Scientific BP2648-250

Zeocin Invitrogen ant-zn-5p

Puromycin Fisher Scientific BP2956-100

96-well microplates Fisher Scientific 12-565-363

Aluminum seals Fisher Scientific 12-565-475

Disposable replicators Fisher Scientific NC9584102

Replication of plates

Prepare target plates by dispensing ~ 160 μL of 2x LB broth (low salt) medium

supplemented with 8% glycerol** and appropriate antibiotic (25 μg/mL Zeocin

and 100 μg/mL carbenicillin).

Prepare source plates

Remove foil seals from the source plates while they are still frozen. This

minimizes cross-contamination. Thaw the source plates with the lid on. Wipe any

condensation underneath the lid with a paper wipe soaked in ethanol.

Replicate

1. Gently place a disposable replicator in the thawed source plate and lightly

move the replicator around inside the well to mix the culture. Make sure to

scrape the bottom of the plate of the well.

2. Gently remove the replicator from the source plate and gently place in the

target plate and mix in the same manner to transfer cells.

3. Dispose of the replicator.

4. Place the lids back on the source plates and target plates.

5. Repeat steps 1-4 until all plates have been replicated.

6. Return the source plates to the -80 °C freezer.

7. Place the inoculated target plates in a 30 °C incubator for 18-19 hours.

Freeze at -80 °C for long-term storage. Avoid long periods of storage at room

temperature or higher in order to control background recombination products.

Protocol II – Plasmid preparation

Culture conditions for individual plasmid preparations

For plasmid preparation, grow all GIPZ shRNA clones at 37 °C in 2x LB broth

(low salt) medium plus 100 μg/mL carbenicillin only.

1. Upon receiving your glycerol stock(s) containing the shRNA of interest,

conrm the clone identity (See: Replication of individual clones) and store

immediately at –80 °C until ready to begin.

2. To prepare plasmid DNA, rst thaw your glycerol stock culture and pulse

vortex to resuspend any E. coli that may have settled to the bottom of

the tube.

3. Take a 10 μL inoculum from the glycerol stock into 3–5 mL of 2x LB broth

(low salt) medium with 100 μg/mL carbenicillin. Return the glycerol stock(s)

to –80 °C.

Dilute the starter culture 1:500–1:1000 into the larger volume.

4. Incubate at 37 °C for 18–19 hours with vigorous shaking.

Due to the tendency of viral vectors to recombine, we

recommend keeping the incubation times as short as

possible and avoid subculturing. Return to your glycerol

stock of your pure culture (see Replication of individual

clones) for each plasmid preparation.

If a large culture volume is desired, incubate the 3-5 mL culture

for 8 hours at 37 ˚C with shaking and use as a starter inoculum.

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Protocol IV – Transfection

Quantities and volumes should be scaled-up according to the number of

cells/wells to be transfected (Table 5). This example is for 24-well plate format.

1. In each well, seed ~ 5 × 10

adherent cells or ~ 5 × 10

suspension cells in

0.5 mL of growth medium 24 hours prior to transfection.

For the library collection, we use the above 96-well bio-

block plasmid preparation protocol in conjunction with

a Qiagen

™

Turbo

™

Kit (Cat #27191). We use 2 bio-blocks

combined. Do not perform the optional wash and elute the

DNA in molecular grade water.

5. Pellet the culture and begin preparation of plasmid DNA. Plasmid DNA

can be isolated using Thermo Scientic

™

GeneJET

™

Plasmid Miniprep Kit

(Cat #K0502) or similar.

6. Run 0.2–1 μg of the plasmid DNA on a 1% agarose gel. pGIPZ with shRNA

is 11774 bp.

Culture conditions for 96-well bio-block plasmid preparation

Inoculate a 96-well bio-block containing 1 mL per well of 2x LB broth (low

salt) medium with 100 μg/mL carbenicillin with 1 µL of the glycerol stock

culture. Incubate at 37 °C with shaking (~ 170–200 rpm). We have observed

that incubation times between 18–19 hours produce good plasmid yield.

For plasmid preparation, follow the protocols recommended by the plasmid

isolation kit manufacturer.

Due to the tendency of viral vectors to recombine, we

recommend keeping the incubation times as short as

possible and avoid subculturing. Return to your original

glycerol stock of your pure culture (see Replication of

individual clones) for each plasmid preparation.

The recommended conﬂuency for adherent cells on the

day of transfection is 70-90%. Suspension cells should be

in logarithmic growth phase at the time of transfection.

2. Dilute 1 µg of DNA in 50 µL of DMEM or other serum-free growth medium.

3. Gently mix DharmaFECT kb transfection reagent and add 3 µL to the diluted

DNA. Mix immediately by pipetting.

4. Incubate 10 minutes at room temperature. Remove medium from

wells and replace with 0.45 mL fresh growth medium.

5. Gently add 50 µL of the DharmaFECT kb reagent/DNA mixture to each well.

6. Gently rock the plate to achieve even distribution of the complexes.

7. Incubate at 37 °C in a CO

incubator.

8. Analyze transgene expression 24-48 hours later. For stable transfection,

cells should be grown in selective medium for 10-15 days (see Protocol V –

Puromycin Selection).

Component Amount

Water, nuclease-free (Cat #R0581) X μL

10x FastDigest

™

buffer 2 μL

DNA sample (up to 1 µg) in water X μL

FastDigest enzyme 1 μL

Final Volume 20 μL

Table 4. Restriction digest components.

1 2 3

5 6

Lane 1 - 10 kb molecular weight ladder (10 kb, 7 kb, 5 kb, 4 kb, 3 kb, 2.5 kb,

2 kb, 1.5 kb, 1 kb)

Lane 2 - Uncut pGIPZ vector

Lane 3 - KpnI digested pGIPZ produces two bands at 1917 bp and 9857 bp.

Lane 4 - SacII digest produces three bands at 1345 bp, 2502 bp and 7927 bp

Lane 5 - SalI produces three bands at 2188 bp, 4465 bp and 5121 bp

Lane 6 - XhoI, NotI double digest produces two bands at 1291 bp & 10,483 bp

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Protocol III – restriction digest

The following is a sample protocol for restriction enzyme digestion using

Thermo Scientic

™

FastDigest

™

Restriction Enzymes KpnI (Cat #FD0524), SalI (Cat

#FD0644), XhoI (Cat #FD0694) and/or NotI (Cat #FD0594) and Thermo Scientic

™

Restriction Enzyme

™

SalI (Cat #ER0205) for diagnostic quality control of pGIPZ

lentiviral vectors.

1. Add the following components (Table 4), in the order stated, to a sterile

PCR thin-wall tube.

2. Mix gently by pipetting.

3. Incubate in a thermal cycler at 37 °C for 5 minutes for FastDigest enzymes

or as suggested by the manufacturer.

4. Load the gel with 10 µL of each of the digested samples, KpnI, SacII, SalI,

XhoI and/or NotI on a 1% agarose gel. Run uncut sample alongside the

digested samples. (Figure 5).

Prepare immediately prior to transfection. We recommend

starting with 1 µg of DNA and 3 µL of DharmaFECT kb

reagent per well in a 24-well plate (see scale-up Table 5).

Subsequent optimization may further increase transfection

efﬁciency depending on the cell line and transgene used.

The transfection efficiency with DharmaFECT kb

transfection reagent (Horizon, Cat #T-2006-01) is equally

high in the presence of serum. This is not the case with

other transfection reagents.

Tissue culture vessel

Growth area,

/well

Volume of

medium, mL

Adherent (suspension) cells to seed

the day before transfection*

Amount of DNA Volume of DharmaFECT kb, μL

µg** µL*** Recommended Range

96-well plate 0.3 0.1 0.5-1.2 × 10

(2.0 × 10

) 0.2 10 0.6 0.4-1.0

48-well plate 0.7 0.25 1.0-3.0 × 10

(5.0 × 10

) 0.5 25 1.5 0.8-2.2

24-well plate 2.0 0.5 2.0-6.0 × 10

(1.0 × 10

) 1.0 50 3.0 2.0-5.0

12-well plate 4.0 1.0 0.4-1.2 × 10

(2.0 × 10

) 2.0 100 6.0 3.9-9.0

6-well plate 9.5 2.0 0.8-2.4 × 10

(4.0 × 10

) 4.0 200 9.0 6.0-12.0

60 mm plate 20 3.0 2.0-6.3 × 10

(1.0 × 10

) 6.0 300 18.0 12.0-24.0

* These numbers were determined using HEK293T and U2OS cells. Actual values depend on the cell type.

** Amount of DNA and DharmaFECT kb transfection reagent used may require optimization.

*** The volume of DNA should be 1/10 of the volume of the culture medium used for dilution of the DNA.

Table 3. Scale-up ratios for transfection of adherent and suspensioncells with DharmaFECT kb transfection reagent

Protocol VII – Titering

Viral titering

Follow the procedure below to determine the titer of your lentiviral stock

using the mammalian cell line of choice IF YOU HAVE PRODUCED VIRAL

PARTICLES YOURSELF. This protocol uses the HEK293T cell line that is

available as part of our Trans-Lentiviral shRNA Packaging Kit (Cat #TLP5918).

You can use a standard HEK293T cell line as an alternative.

1. The day before transduction, seed a 24-well tissue culture plate

with HEK293T Cells at 5 × 10

cells per well in DMEM (10% FBS,

1% pen-strep).

2. Make dilutions of the viral stock in a round bottom 96-well plate

using serum-free media. Utilize the plate as shown in (Figure 6)

using one row for each virus stock to be tested. Use the procedure below

(starting at step 4) for dilution of the viral stocks. The goal is to produce a

series of 5-fold dilutions to reach a final dilution of 390,625-fold.

3. Add 80 µL of serum-free media to each well.

4. Add 20 µL of thawed virus stock to each corresponding well in

column 1 (five-fold dilution).

If you have generated a lentiviral stock of the expression

control (such as GIPZ non-silencing construct), we

recommend titering this stock as well.

The following day, each well should be no more

than 40-50% confluent.

Pipette contents of well up and down 10-15 times. Discard

pipette tip.

Figure 6. Five-fold serial dilutions of virus stock.

Virus stock 1

Virus stock 2

Virus stock 3

Virus stock 4

Dilution Plate

1 2345678910 11 12

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Protocol V – Puromycin selection

Determining antibiotic dose response (Kill curve)

In order to generate stable cell lines expressing the transgene of interest, it

is important to determine the minimum amount of antibiotic required to kill

non-transfected cells. A simple procedure to test this is as follows:

1. Day 1: Using the same cell type and relative cell densities to be used in

subsequent transfection or transduction procedures, plate cells

and culture overnight.

2. Day 2: Replace complete growth medium with growth medium

supplemented with a range of puromycin concentrations (0-15 μg/mL),

including untreated control cells with no antibiotic added.

3. Day 4: Refresh medium and assess viability.

4. Replace medium with fresh medium supplemented with the appropriate

concentration of puromycin every 2-3 days depending on the growth

of cells.

5. Examine cells daily and identify the minimal concentration of antibiotic

that efficiently kills all non-transfected/transduced cells between

3-6 days following addition of puromycin.

Puromycin selection

If adding antibiotic for selection, use the appropriate concentration as

determined based on the above kill curve.

1. Add medium containing antibiotic 24 or hours post-transfection or

post-transduction, respectively, to begin selection

2. Cells can be harvested for transgene expression 24-72 hours after

starting selection.

3. If longer selection is required for cells to be confluent, replace selective

medium approximately every 2-3 days.

4. Monitor the cells daily and observe the percentage of surviving cells.

Cells surviving selection will be expressing the transgene.

5. If generating stable cell lines (optional), select and grow for 10-15 days.

6. Once non-transfected cells are eliminated and/or you have selected for

stably transfected cell lines if desired, you can proceed to assay for target

gene expression. RT-qPCR, Western blot analysis or other appropriate

functional assay can be used; compare treated samples to untreated,

reporter alone, non-silencing control, or other controls as appropriate.

Protocol VI – Packaging lentivirus

The pGIPZ vector is tat dependant, so you must use a packaging system

that expresses the tat gene. For packaging our lentiviral shRNA constructs,

we recommend the Trans-Lentiviral shRNA Packaging Kit (Cat #TLP5912

or TLP5917). The Trans-Lentiviral shRNA Packaging System allows creation

of a replication-incompetent, HIV-1-based lentivirus which can be used to

deliver and express your shRNA of interest in either dividing or non-dividing

mammalian cells. The Trans-Lentiviral shRNA Packaging System uses a

replication-incompetent lentivirus based on the trans-lentiviral system

developed by Kappes (Kappes 2001). For protocols and information on

packaging pGIPZ with our Trans-Lentiviral shRNA Packaging System, please

see the product manual available on our website.

It is important to wait at least 24 hours after transfection

before beginning selection.

Once you have generated a lentiviral stock with a suitable titer, you are ready to

transduce the lentiviral vector into the mammalian cell line of choice and assay

for gene silencing

Multiplicity of infection (MOI)

To obtain optimal silencing of your shRNA, you will need to transduce the

lentiviral vector into your mammalian cell line of choice using a suitable MOI.

MOI is defined as the number of transducing units per cell.

Determining the optimal MOI

A number of factors can influence determination of an optimal MOI including

the nature of your mammalian cell (actively dividing versus non-dividing), its

transduction efficiency, your application of interest, and the nature of your gene

of interest. If you are transducing your lentiviral construct into the mammalian cell

line of choice for the first time, after you have titered the lentiviral particles, we

recommend using a range of MOIs (for example, 0, 0.5, 1, 2, 5, 10, 20) to determine

the MOI required to obtain optimal expression for your particular application.

It should be noted that to achieve single copy knockdown, an MOI of 0.3 is

generally used, as less than 4% of your cells will have more than one insert.

Protocol VIII – Transduction

Transduction of target cells

The protocol below is optimized for transduction of the lentiviral particles into

HEK293T, OVCAR-8 or MCF7 cells in a 24-well plate using serum-free media. If a

different culture dish is used, adjust the number of cells, volumes, and reagent

quantities in proportion to the change in surface area (Table 7). It is strongly

recommended that you optimize transduction conditions to suit your target cell

line to provide for the highest transduction efficiency possible.

It is preferable that transduction be carried out in medium that is serum

free and antibiotic free. A reduction in transduction efficiency occurs in the

presence of serum; however it is possible to carry out successful transductions

with serum present; you will have to optimize the protocol according to your

cells of interest.

5. With new pipette tips, transfer 20 µL from each well of column 1

to the corresponding well in column 2.

6. With new pipette tips, transfer 20 µL from each well of column 2 to the

corresponding well in column 3.

7. Repeat transfers of 20 µL from columns 3 through 8, pipetting up and

down 10-15 times and changing pipette tips between each dilution.

8. Label 24-well plate as shown in (Figure 7) using one row for each virus

stock to be tested.

9. Remove culture media from the cells in the 24-well plate.

10. Add 225 µL of serum-free media to each well.

11. Transduce cells by adding 25 µL of diluted virus from the original

96-well plate (Figure 6) to a well on the 24-well destination plate

(Figure 7) containing the cells. For example, transfer 25 µL from well

A2 of the 96-well plate into well A1 in the 24-well plate (Table 6).

12. Incubate transduced cultures at 37 °C for 4 hours.

13. Remove transduction mix from cultures and add 1 mL of DMEM

(10% FBS, 1% Pen-Strep).

14. Culture cells for 48 hours.

15. Count the TurboGFP expressing cells or colonies of cells (Figure 8).

16. Transducing units per mL (TU/mL) can be determined using the

following formula:

# of TurboGFP positive colonies counted × dilution factor × 40 = # TU/mL:

Example: 55 TurboGFP positive colonies counted in well A3

55 (TurboGFP positive colonies) × 625 (dilution factor) × 40 = 1.38 ×

TU/mL

Pipette up and down 10-15 times and discard pipette tip.

It is strongly recommended that you use a high quality

multichannel pipettor when performing multiple dilutions.

Pre-incubate the dilutions of the virus stock for 5 minutes

at room temperature.

Count each multi-cell colony as 1 transduced cell, as the

cells will be dividing over the 48 hour culture period. Figure

8 illustrates this principle of cell counting.

1 2 3 4 5 6

Virus stock 1

Virus stock 2

Virus stock 3

Virus stock 4

Figure 7. Twenty four well tissue culture plate, seeded with HEK293T cells, used

to titer the virus.

Figure 8. Examples of individual colonies.

A B

Well (Row A, B, C, or D)

Volume diluted

virus used

Dilution factor

Originating

(96-well plate)

Destination

(24-well plate)

A1 25 μL 5 *

A2 A1 25 μL 25

A3 A2 25 μL 125

A4 A3 25 μL 625

A5 A4 25 μL 3125

A6 A5 25 μL 15625

A7 A6 25 μL 78125

A8 25 μL 390625 *

Table 6. Example of set up for dilutions.

*Please note that when expecting very high or very low titers, it would be advisable to

include either well 8 or well 1 respectively.

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1. On day 0, plate 5 × 10

cells per well in a 24-well plate. Incubate

overnight. You will be using full growth medium (with serum) at

this stage.

2. The next day (day 1), remove the medium and add an appropriate

amount of the virus to acheive the MOI you wish to use. Set up

all desired experiments and controls in a similar fashion. Bring

the total volume of liquid up so that it just covers the cells

efficiently with serum-free media (see Table 7 for guidelines).

If you are using concentrated virus you are likely to use a small

volume of virus if you are using unconcentrated virus, you will

find you need more virus volume.

3. Approximately 4-6 hours post-transduction, add an additional 1 mL

of full medium (serum plus Pen-Strep if you are using it) to your cells

and incubate overnight.

At 48 hours post-transduction examine the cells microscopically for

the presence of reporter gene (TurboGFP) expression as this will be

your first indication as to the efficiency of your transduction.

a. If adding puromycin, use the appropriate concentration as

determined based on the kill curve (see Protocol V). Incubate cells

with the selection medium.

b. Approximately every 2-3 days replace with freshly prepared

selective medium.

c. Monitor the cells daily and observe the percentage of surviving

cells. Once the non-transduced control cells are dead, the surviving

cells in the transduced wells will be expressing the shRNA

Optimum effectiveness of the puromycin selection should be

reached in 4-6 days with puromycin dependent upon the

concentration of puromycin chosen from the kill curve.

We have experienced low toxicity with transduction in the

cell lines tested, therefore removal of virus is not required

for many cell lines. In our experience, higher transduction

efficiencies have been achieved if the virus is not removed

after 6 hours. However, if toxicity is a problem, aspirate

the mixture after 4-6 hours and replace with fresh growth

medium. Additionally, fresh growth medium should be

replenished as required for continued cell growth.

The higher the MOI you have chosen, the more copies

of the shRNA and puromycin resistance gene you will

have per cell. When selecting with puromycin, it is worth

remembering that at higher MOIs, cells containing multiple

copies of the resistance gene can withstand higher

puromycin concentrations than those at lower MOIs. Adjust

the concentration of puromycin to a level that will select

for the population of transduced cells you require for your

application without going below the minimum antibiotic

concentration you have established in your kill curve.

Once your transduction efficiency is at an acceptable level (with or without

puromycin selection performed post-transduction), you can proceed to assay

cells for reduction in gene expression or fluorescent reporter activity by reverse

transcription quantitative/real-time PCR (RT-qPCR), Western blot analysis or

other appropriate functional assay. Compare target gene to untreated, reporter

alone (empty vector), non-silencing shRNA, or other negative controls.

Protocol IX – Determining relative

transduction eciency

Follow the procedure below to determine the relative transduction efficiency

of purchased GIPZ lentiviral particles (Cat #VGH5518, VGM5520, VGH5526). This

protocol should only be used with purchased GIPZ shRNA individual clones in

viral particle format.

Prior to transducing with purchased GIPZ shRNA individual clones in viral

particle format, we recommend determining the relative transduction efficiency

of your cell type. Lentiviral titers provided with purchased GIPZ lentiviral

particles have been calculated by transducing HEK293T cells. Transduction

efficiencies vary significantly by cell type.

The relative transduction efficiency of your cells may be estimated by

determining the functional titer of a control virus such as GIPZ Non-silencing

control viral particles (Cat #RHS4348) in your cells of interest.

Follow the procedure below to determine the functional titer of the GIPZ

Non-silencing control shRNA viral stock in the mammalian cell line of your

choice. The following conditions have been optimized for transduction of

HEK293T cells. When determining the relative transduction efficiency of

your cell type, use the transduction conditions that have been optimized

for your cells of interest.

1. The day before transduction (day 0), seed a 24-well tissue culture plate

with your cells at 5 × 10

cells per well in their respective medium.

2. Make dilutions of the Non-silencing control shRNA viral stock in a round

bottom 96-well plate using serum-free medium. Utilize the plate as shown

in Figure 9 with one row for each replicate (we recommend performing

at least two replicates). Use the procedure below for dilution of the viral

stock. The goal is to produce a series of five-fold dilutions to reach a final

dilution of 390, 625-fold.

Optimal length of incubation from the start of transfection

to analysis is dependent on cell type, gene of interest,

and the stability of the mRNA and/or protein being

analyzed. RT-qPCR generally gives the best indication of

mRNA expression and gene silencing. The use of Western

blot analysis to determine knockdown is dependent on

quantity and quality of the protein, its half-life, and the

sensitivity of the antibody and detection systems used.

The following day, each well should be no more than

40-50% confluent.

When visualizing TurboGFP expression, if less than

90% of all cells are green, it is recommended in these

cases to utilize puromycin selection in order to reduce

background expression of your gene of interest from

untransduced cells.

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Tissue

culture dish

Surface area

per well (cm

)

Suggested total serum-free medium

volume per well (mL)

100 mm 56.0 5.0

60 mm 20.0 2.0

35 mm 8.0 1.0

6-well 9.4 1.0

12-well 3.8 0.5

24-well 1.9 0.25

96-well 0.3 0.1

Ta ble 7. Suggested volumes of media per surface area per well of adherent cells.

3. Add 40 uL of serum-free media to each well in column.

4. Add 80 uL of serum-free media to each well of columns 2-8.

5. Add 10 µL of thawed control shRNA virus stock to each well in column 1

(five-fold dilution).

6. With new pipette tips, transfer 20 µL from each well of column 1 to the

corresponding well in column 2.

Pipette up and down 10-15 times and discard pipette tip.

7. With new pipette tips, transfer 20 µL from each well of column 2 to the

corresponding well in column 3.

Pipette up and down 10-15 times and discard pipette tip.

8. Repeat transfers of 20 µL from columns 3 through 8, pipetting up and

down 10-15 times and changing pipette tips between each dilution.

It is strongly recommended that you use a high-quality

multichannel pipettor when performing multiple dilutions.

Incubate the dilutions of the virus stock for 5 minutes at room

temperature.

9. Label a 24-well plate as shown in Figure 10 using one row for each

replicate.

10. Remove culture medium from the cells in the 24-well plate.

11. Add 225 µL of serum-free medium to each well.

12. Transduce cells by adding 25 µL of diluted control shRNA lentivirus

from the original 96-well plate (Figure 9) to a well on the 24-well

destination plate (Figure 10) containing the cells. For example,

transfer 25 µL from well A2 of the 96-well plate into well A1in the

24-well plate (Table 8).

13. Incubate transduced cultures at 37 °C for 4-6 hours.

If desired, include 8 μg/mL polybrene in the

dilution medium.

Pipette contents of well up and down 10-15 times.

Discard pipette tip.

Post dox 72 hours

14. Add 1 mL of your medium (normal serum concentration).

15. Culture cells for 72 hours.

16. Count the TurboGFP expressing cells or colonies of cells (Figure 11). Count

each multi-cell colony as 1 transduced cell, as the cells will be dividing

over the 72 hour culture period. Figure 11 illustrates this principle of

cell counting. Count the number of TurboGFP expressing colonies in

wells corresponding to at least two viral dilutions.

17. Transducing units per ml (TU/mL) can be determined using the following

formula: # of TurboGFP positive colonies counted × dilution factor × 40 =

# TU/mL

Example: 55 TurboGFP positive colonies counted in well A3 55 (TurboGFP

positive colonies) × 625 (dilution factor) × 40 =

1.38 × 10

TU/mL.

18. The functional titer calculated for your cell line under your experimental

conditions can be used to determine the relative transduction efficiency

of your cell type by using the following formula: Functional titer of Non-

silencing control shRNA virus stock in your cell line ÷ Titer of Non-silencing

control shRNA virus stock as calculated in HEK293T = Relative transduction

efficiency.

For example, if the titer of the Non-silencing control shRNA virus stock in

HEK293T (as provided on the product specification sheet) is 6.9 × 10

TU/mL

and the functional titer of the control shRNA virus stock in your cell line is 1.38

× 10

TU/mL, the relative transduction efficiency of your cell type is 0.2. To

extrapolate the average functional titer of the provided GIPZ viral particles,

multiply the average titer of each plate as provided on the product specification

sheet by the relative transduction efficiency of your cell type. In our example,

if the titer of the GIPZ viral particles in HEK293T cells is 2 × 10

TU/mL and the

relative transduction efficiency of your cell type is 0.2, the extrapolated average

functional titer of that plate your cell type is 4 × 10

TU/mL.

25 μLof diluted virus was added to the cells. This is

1/40th of a mL.

1 2 3 4 5 6

Virus stock 1

Virus stock 2

Virus stock 3

Virus stock 4

Figure 10. Twenty-four well tissue culture plate, seeded

with HEK293T cells, used totiter the virus.

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Figure 9. Five-fold serial dilutions of virus stock.

Virus stock 1

Virus stock 2

Virus stock 3

Virus stock 4

Dilution Plate

1 2345678910 11 12

Figure 11. Examples of individual colonies.

A B

*Please note that when expecting very high or very low titers, it would be

advisable to include either well 8 or well 1 respectively.

Well (row A, B, C, or D)

Volume diluted

virus used

Dilution

factor

Originating

(96-well plate)

Destination

(24-well plate)

A1 25 μL 5 *

A2 A1 25 μL 25

A3 A2 25 μL 125

A4 A3 25 μL 625

A5 A4 25 μL 3125

A6 A5 25 μL 15625

A7 A6 25 μL 78125

A8 25 μL 390625 *

Table 8. Example of set up for dilutions.

Once the relative transduction efficiency of the GIPZ virus has been

established in your cell line, use the optimized transduction conditions

determined in Protocol VIII to transduce your cell line with the purchased

GIPZ shRNA individual clones in viral particle format. If the titer of the non-

silencing control shRNA virus is not satisfactory in your cell line you might

consider choosing a different cell line more permissive to transduction by

lentivirus before proceeding.

Protocol X – PCR

QPCR experimental recommendations

One of the biggest challenges of any qPCR experiment is to obtain

reproducible and reliable data. Due to the sensitivity of this multi-step

technique, care must be taken to ensure results obtained are accurate and

trustworthy (see Bustin et al., 2009).

1. Experimental samples should be run at least in duplicate. It should

be noted that with duplicate experiments it will not be possible to

assign error bars to indicate consistency from experimental sample

to experimental sample. Using triplicate samples or higher will

enable error bars to be assigned indicating the level of experimental

variation.

2. Reverse Transcriptase reactions for cDNA synthesis should always

include a No Template Control (NTC) and No Reverse Transcriptase

(no RT) control to check for reagents contamination and the

presence of contaminating DNA, respectively. Use a robust reverse

transcriptase enzyme for cDNA synthesis such as the Thermo

Scientific

™

Maxima

™

cDNA Synthesis Kit for RT-qPCR (Cat #K1641).

3. We have found that normalizing the RNA concentration prior to

cDNA synthesis will increase consistency downstream.

4. qPCR should be done at least in triplicate. Again, it should be noted

that with duplicate reactions it will not be possible to assign error

bars to indicate the consistency in your qPCR reactions. Using

triplicate samples or higher will enable error bars to be assigned

indicating the level of variation between qPCR reactions. Use

validated primer sets for SYBR-based assays or primers/probe for

probe-based assays.

5. Make sure the mRNA you are using as your internal reference control

for qPCR is expressed at a level higher than your target gene's

message.

6. Use only high-quality calibrated pipettes, in conjunction with well

fitting barrier tips.

7. When pipetting, take the time to visually inspect the fluid in the

pipette tip(s) for accuracy and lack of bubbles, especially when using

a multi-channel pipette.

8. Be sure to spin your qPCR plate prior to loading in the real-time

instrument in order to collect the sample at the bottom of the well

and eliminate any bubbles that may have developed.

9. With regard to knockdown experiments using shRNA, it is vitally

important that you greatly reduce if not eliminate entirely those

cells which are not transduced or transfected from the population.

This can be done in several ways: increase the efficiency of your

transfection, use a higher mulitplicity of infection (MOI) for

your transduction, utilize the puromycin selection marker and

select against those cells that do not contain the shRNA or utilize

fluorescent sorting to select against those cells that do not contain

the shRNA.

10. Always utilize the non-silencing control as a reference for target gene

expression, as opposed to an untreated sample. The non-silencing

treated samples will most accurately reproduce the conditions in your

experimental samples. The non-silencing best controls for changes in

qPCR internal reference gene expression.

11. You may also use an untreated sample to indicate substantial

changes in target gene expression as seen in the non-silencing control

due to generic consequences of viral transduction and transgene

expression. However, it should be noted that small changes in

expression levels between an untreated sample and the non-silencing

control are to be expected.

12. Cq values greater than 35 should be avoided as they tend to be

variable. Samples with such high Cq values should be repeated at

higher cDNA concentrations and with a lower expressing qPCR internal

reference control (such as TBP).

13. Cq values less than 11 for the qPCR internal reference control should be

avoided as it is difficult to determine a proper background subtraction

using these values. If this occurs, use Cq values from both your internal

reference control as well as your experimental target to determine an

optimum cDNA concentration.

14. It may be necessary to change internal reference controls if conditions

in steps 12 and 13 cannot be simultaneously met.

100%

113%

88%

93%

120%

140%

Control GAPDH EG5 PP1B

100%

80%

60%

40%

20%

Residual Gene

Activity

Figure 12. Non-silencing lentiviral shRNA control does not knockdown common

endogenous genes. The above data represents the baseline amount of GAPDH, EG5 or

PP1B mRNA set at 100% in the control. The relative amounts of each of these mRNAs are

then represented after treatment with non-silencing shRNA of these genes.

Figure 13. HEK293T cells were transduced with lentiviral particles expressing GAPDH

or Non-silencing shRNA at variable MOIs ranging from 9-48. The graph depicts the

residual levels of GAPDH relative to Non-silencing control.

120%

Untrans-

duced

Control

NS#1 NS#1 NS#1 GAPDH GAPDH GAPDH

100%

80%

60%

40%

20%

12%

28%

34%

100%100%100%100%

Residual GAPDH

Expression

horizondiscovery.com

Figure 15. The characteristic phenotype observed by the targeting of the EG5 (KIF11)

gene results in the formation of half spindles, mitotic arrest and monoastral microtubular

arrays (green, see the cell on the left). By contrast, normal cells show bipolar spindles

and microtubule networks in mitosis and in interphase (see the cell on the right). The

comparative expression of EG5 (red) between the cell on the left and the right shows

the extensive knockdown of EG5 in the cell displaying the phenotype (left). The cells

were visualized at 100x magnification using aLeica DMIRB fluorescence microscope.

HEK293T cells were stained for tubulin (anti-tubulin, green), DNA (DAPI, blue) and EG5

(anti-EG5, red).

OVERLAY ANTI-TUBULIN

ANTI-EG5 DAPI

Related Reagents Dharmacon Cat #

GAPDH Verified Positive Control* RHS4371

EG5 Verified Positive control* RHS4480

Non-silencing Verified Negative Control* RHS4346

DharmaFECT kb transfection reagent 1 mL 2006-01

GIPZ shRNA Empty Vector RHS4349

Trans-Lentiviral shRNA Packaging System TLP5912

Trans-Lentiviral shRNA Packaging System with HEK293T Cells TLP5917

Table 9. Related Reagents.

*These items also available in the GIPZ lentiviral transfection starter kit (Cat #RHS11851).

Controls and validation

RNAintro shRNA starter kits

The use of vector-based RNAi for gene silencing is a powerful and versatile tool.

Successful gene silencing in vitro is dependent on several variables including 1)

The target cell line being studied, 2) transfection and transduction efficiency,

3) abundance of the mRNA or protein of interest in the target cell line, 4) half

life of the protein, and 5) robust experimental protocols. For all these reasons,

it is important to run controlled experiments where the transfection and

transduction efficiencies are as high as possible and measurable.

Controls are a critical part of a gene silencing experiment. They enable

accurate representation of knockdown data and provide confidence in the

specificity of the response. Changes in the mRNA or protein levels in cells

treated with negative or non-silencing controls reflect non-specific responses

in cells and can be used as a baseline against which specific knockdown

can be measured. Positive controls are useful to demonstrate that your

experimental system is functional.

Controls

The EG5 and GAPDH GIPZ lentiviral shRNA vectors have been validated as

positive controls for RNAi experiments performed using the GIPZ shRNA-

containing lentiviral vectors. These shRNA have been tested in transduction

based experiments and have shown efficient knockdown at both mRNA and

protein levels. The EG5 control has been validated to knockdown human EG5 by

RT-qPCR (Figure 14 and 15) and in situ hybridization of cells in tissue culture. The

GAPDH control has been validated to knockdown human and mouse GAPDH

by RT-qPCR (Figure 13). The GIPZ Non-silencing lentiviral shRNA vector has been

validated as a negative control for RNAi experiments performed using the GIPZ

shRNA-containing lentiviral vectors (Figure 12).

Frequently Asked Questions (FAQs)

What clones are part of my library collection?

A USB containing the data for this collection will be shipped with each

collection. This file contains the location and accession number for each

construct in the collection.

Where can I find the sequence of an individual shRNA construct?

If you are looking for the sequence of an individual shRNA construct, you can

search for the clone on our website (horizondiscovery.com). Enter the catalog

number or clone ID of your construct into the search at the top of the page. You

should see your product in the catalog number section of the results. Click on

the plus sign to expand the details for this clone and select the Sequence tab.

Which antibiotic should I use?

You should grow all GIPZ shRNA constructs in 2x LB broth medium with both

25 μg/mL zeocin and 100 μg/mL carbenicillin for archive replication. For

plasmid preparations, grow the constructs in 2x LB broth medium containing

only 100 ug/mL carbenicillin.

What packaging cell line should I use for making lentivirus?

The GIPZ shRNA vector is tat dependant, so a packaging system that

expresses the tat gene. For packaging our lentiviral shRNA constructs, we

recommend the Trans-Lentiviral shRNA Packaging Kit (TLP5912, TLP5917). The

Trans-Lentiviral Packaging Kit allows creation of a replication-incompetent

(Shimada, et al. 1995), HIV-1-based lentivirus which can be used to deliver

and express your gene or shRNA of interest in either dividing or non-dividing

mammalian cells. The Trans-Lentiviral Packaging Kit uses a replication-

incompetent lentivirus based on the trans-lentiviral system developed

by Kappes (Kappes and Wu et al. 2001). For protocols and information on

packaging GIPZ shRNA with our Trans-Lentiviral shRNA Packaging Kit, please

see the product manual available at here.

Can I use any 2

generation packaging system with the pGIPZ vector?

The pGIPZ vector is tat dependant, so you must use a packaging system that

expresses the tat gene.

What does the number 40 refer to in the formula for the calculation of titer?

The titer units are given in transducing units (TU) per mL, so the number 40 is

used to convert the 25 µL used in the titration (“volume of diluted virus used”,

Table 6) to one milliliter.

What is the sequencing primer for pGIPZ?

The pGIPZ sequencing primer is 5' - GCATTAAAGCAGCGTATC - 3'

Note: The binding site lies from base 5820-5842 and runs in the reverse

complement direction. The melting temperature of this 18mer = 52.7 °C.

Where do you purchase puromycin?

We purchase puromycin from Fisher Scientific Cellgro (Cat #BP2956-100).

How many transfections are available in each volume size of

DharmaFECT kb transfection reagent?

The number of transfections that can be performed depends on the size of the

culture dish used.

120%

NS#1

MOI 3.5

NS#1

MOI 8.5

NS#1

MOI 17

EG5

MOI 3.5

EG5

MOI 8.5

EG5

MOI 17

100%

80%

60%

40%

20%

17%

18%

29%

100%100%100%

Residual EG5

Expression

Figure 14. HEK293T cells were transduced with lentiviral particles expressing EG5 or

non-silencing shRNA at MOIs of 3.5, 8.5 and 17. The graph depicts the residual levels of

EG5 relative to its non-silencing control.

Troubleshooting

For help with transfection or transduction of your lentiviral constructs, please

email technical support at technical@horizondiscovery.com with the answers

to the questions below, your sales order or purchase order number and the

catalog number or clone ID of the construct with which you are having trouble.

1. Are you using direct transfection or transduction into your cell line?

2. What was the 260/280 ratio of DNA? Over 1.8?

3. What was the transfection efficiency if you used direct transfection?

What transfection reagent was used?

4. Were positive and negative knockdown controls used (such as our

GAPDH or EG5 validated positive controls and the validated non-

silencing negative control)?

5. What were the results of the controlled experiments?

6. How was knockdown measured (for example real-time RT-qPCR or

western blot analysis)?

7. What is the abundance and the half-life of the protein? Does the protein

have many isoforms?

8. What packaging cell line was used if you are using transduction rather

than transfection?

9. What was your viral titer?

10. What was your MOI?

11. Did you maintain the cells in puromycin selection media after

transfection or transduction?

12. How much time elapsed from transfection/transduction to

puromycin selection?

If transfection into your cell line is unsuccessful, you may need to

consider the following list of factors influencing successful transfection.

1. Concentration and purity of plasmid DNA and nucleic acids—determine

the concentration of your DNA using 260 nm absorbance. Avoid

cytotoxic effects by using pure preparations of nucleic acids.

2. Insufficient mixing of transfection reagent or transfection complexes.

3. Presence of antibiotics in transfection medium—the presence of antibiotics

do not interfere with both DNA/DharmaFECT kb complex formation and cell

transfection. This is not the case for other transfection reagents.

4. Cell history, density, and passage number—it is very important to use

healthy cells that are regularly passaged and in growth phase. The highest

transfection efficiencies are achieved if cells are plated the day before;

however, adequate time should be given to allow the cells to recover from

the passaging (generally > 12 hours). Plate cells at a consistent density to

minimize experimental variation. If transfection efficiencies are low or

reduction occurs over time, thawing a new batch of cells or using cells with

a lower passage number may improve the results.

If transduction into your cell line is unsuccessful, you may need to

consider the following list of factors influencing successful transduction.

1. Transduction efficiency is integrally related to the quality and the

quantity of the virus you have produced. Factors to consider when

transducing include MOI (related to accurate titer in the target cell line),

the presence of serum in the media, the use of polybrene in the media,

length of exposure to virus, and viral toxicity to your particular cells.

2. High quality transfer vector DNA and the appropriate and efficient viral

packaging are required to make high quality virus able to transduce

cells effectively.

3. See also suggestions 3–5 for factors influencing successful

transfection (above).

References

Cited References and additional suggested reading

1. Bartel, D. P. (2004). microRNAs: genomics, biogenesis, mechanism, and

function. Cell 116(2): 281- 97.

2. Boden, D., O. Pusch, et al. (2004). Enhanced gene silencing of HIV-1 specific

siRNA using microRNA designed hairpins Nucleic Acids Res 32(3): 115 4 -8.

3. Chendrimada, T. P., R. I. Gregory, et al. (2005). TRBP recruits the Dicer

complex to Ago2 for microRNA processing and gene silencing. Nature

436(7051): 740-4.

4. Cleary, M. A., K. Kilian, et al. (2004). Production of complex nucleic acid

libraries using highly parallel in situ oligonucleotide synthesis.

Nat Methods 1(3): 241-8.

5. Cullen, B. R. (2004). Transcription and processing of human microRNA

precursors. Mol Cell 16(6): 861-5.

6. Cullen, B. R. (2005). RNAi the natural way. Nat Genet 37(11): 1163-5.

7. Dickins, R. A., M. T. Hemann, et al. (2005). Probing tumor phenotypes using

stable and regulated synthetic microRNA precursors. Nat Genet 37(11):

1289 -95.

8. Editors of Nature Cell Biology (2003). Whither RNAi? Nat Cell Biol 5(6):

489-90.

9. Elbashir, S. M., J. Harborth, et al. (2001). Duplexes of 21-nucleotide

RNAs mediate RNA interference in cultured mammalian cells. Nature

411(6836): 494-8.

10. Fire, A., S. Xu, et al. (1998). Potent and specific genetic interference by

double-stranded RNA in Caenorhabditis elegans. Nature 391(6669):

806 -11.

11. Gregory, R. I., T. P. Chendrimada, et al. (2005). Human RISC couples

microRNA biogenesis and posttranscriptional gene silencing.

Cell 123(4): 631-40

12. Kappes, J. C. and X. Wu (2001). Safety considerations in vector

development. Somat Cell Mol Genet 26(1-6):147-58.

13. Kappes, J. C., X. Wu, et al. (2003). Production of trans-lentiviral vector

with predictable safety. Methods Mol Med 76: 449-65.

14. Paddison, P. J., J. M. Silva, et al. (2004). A resource for large-scale

RNA-interference-based screens in mammals. Nature 428(6981): 427-31.

15. Shimada, T., et.al. (1995). Development of vectors utilized for gene

therapy for AIDS. AIDS 4.

16. Silva, J. M., M. Z. Li, et al. (2005). Second-generation shRNA libraries

covering the mouse and human genomes. Nat Genet 37(11): 1281-8.

Lable licenses

The shRNA Products, use and applications, are covered by pending and

issued patents. Certain Label licenses govern the use of the products, these can

be found at dharmacon-licensing-statements. It is each Buyer’s responsibility to

determine which intellectual property rights held by third parties may restrict

the use of Products for a particular application. Please review the Label Licenses

governing all use of the shRNA and gene expression products.

To ﬁnd the contact information in your country for your technology of interest, please visit us at

horizondiscovery.com/contact-us

Horizon Discovery, 8100 Cambridge Research Park, Waterbeach, Cambridge, CB25 9TL, United Kingdom

Dharmacon Inc. TURBOGFP is a trademark of Evrogen JS . HXB2 is a trademark of GenBank. ZEOCIN, FASTDIGEST, GENEJET, and MAXIMA are trademarks of Thermo Fisher Scientiﬁc, Inc

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